Biopolymers such as peptides, proteins, nucleic acids (RNA or DNA), oligosaccharides, or complexes thereof are commonly analyzed by gel electrophoresis. Usually samples of interest are loaded in a matrix such as a polyacrylamide gel and exposed to an electric field which causes various components in the sample to migrate and separate into distinct bands according to the molecular weight, net charge, size, and other physical and chemical properties of the molecules and the pore size of the matrix. After electrophoresis, different biopolymers embedded at different locations on the matrix can be further characterized by their interactions with or bindings to one or more binding agents, such as staining reagents.
Numerous methods and reagents have been developed to visualize or detect the biopolymers of interest within a matrix such as a gel. These include staining reagents that can be classified into five classes. The first class of staining reagents includes organic dyes that bind to biopolymers, such as Coomassie Blue dyes that stain proteins and make the protein bands blue, which can be subsequently visualized by naked eyes. The second class of staining reagents includes fluorescent dyes that bind to biopolymers, such as ethidium bromide that stains DNA or RNA and makes the stained DNA or RNA bands red when shined with UV light. The third class of staining reagents includes silver staining reagents. The fourth class of staining reagents includes staining reagents that stain the background, which are also called negative staining reagents. The fifth class of staining reagents includes biological molecules and their derivatives, such as antibodies and antibody-based reagents that bind to antigens, which are also called immunostaining reagents, or labeled polynucleotide that binds to complementary DNA or RNA. The second and third classes of staining reagents were developed to increase the sensitivity over that achieved with the organic dyes of the first class.
Coomassie Blue staining was introduced in early 1960s by Fazekas, et al. as a method for visualizing proteins in the gels (Biochim. Biophys. Acta 71:377, 1963). In addition to Coomassie Blue, other organic dyes such as Amido black, Ponceaus S, Fast green FCF, zincon, Eriochrome black T, etc., have also been used to stain proteins. However, Coomassie Blue staining is still widely used and remains the most commonly used general method for protein staining and detection.
Typically, a conventional method for Coomassie Blue staining of proteins embedded in a gel comprises the following steps: (i) fixing the gel in Fixing Solution for 1 hour (hr) with gentle agitation; (ii) staining the gel from Step (i) in Staining Solution for 1 hr with gentle agitation; and (iii) destaining the gel from Step (ii) in Destaining Solution. The Destaining Solution is replaced several times during the destaining step until the background of the gel is fully destained, which usually takes 2 to 3 hours or even overnight.
Staining proteins with organic dyes is relatively inexpensive, takes less time than silver staining, but it still takes a few hours or even overnight. For example, the above described conventional method of Coomassie Blue staining takes about 3 hours or more with at least 3 steps, thus is time consuming and cumbersome. Improvements have been made in Coomassie Blue protein staining methods, e.g., by using new staining reagents such as Colloidal Coomassie Blue, or by performing the incubation in a microwave to enhance staining and reduce the incubation time. However, the three basic steps of fixing, staining and destaining are still necessary to obtain satisfactory results.
Immunostaining of antigens in a matrix, such as a polyacrylamide gel, also takes many steps and multiple solutions. Each step may take minutes to hours to complete, in part because the staining reagents, e.g., antibodies and antibody-based reagents, diffuse into the matrix slowly. Similarly, the hybridization of a polynucleotide with another polynucleotide, such as the Southern or Northern blotting, is also time consuming.
Thus, there is still a need for a simple and rapid process for staining a biopolymer, such as a peptide, a protein, an RNA, a DNA, an oligosaccharide or a complex thereof. Embodiments of the present invention relate to such a process for staining, thus detection, of biopolymers embedded in a matrix with reduced time and costs.